Splicing of pre-mRNAs provides a major source of transcript diversity for cell differentiation and development. The process of splicing requires a splicing machine known as the spliceosome that comprises ~100 proteins and five small nuclear (sn)RNAs. The early stage of spliceosome assembly on pre-mRNA splice sites is a key regulated step that often goes awry in human genetic diseases and cancers. Yet, exactly how the spliceosome selects and excises the correct splice sites from amidst thousands of competing pre-mRNA sequences remains poorly understood at the molecular level. The overall goal of this project is to understand how an essential complex comprising U2AF65, U2AF35, and SF1 proteins can accurately target 3' splice sites, leading to spliceosome assembly. Specific aims of this proposal will test the following central hypotheses concerning the critical early stages of pre-mRNA splicing: Hypothesis #1: Our 'panoptic' understanding of U2AF65 recognition of 3' splice site RNAs - emerging from our new structures of intact U2AF65 as well as past work on core domains- can be used to understand pre-mRNA splice site mutations from specific human diseases. Hypothesis #2: We will expand our prior results and preliminary data to test the spliceosome subunit SF3b155 as a molecular hub coordinating phosphorylation-sensitive assemblies comprising U2AF65, the cancer-related factor paralogue CAPERa, and the p14 subunit, which in turn contacts the branch-site nucleophile of the spliceosome. Hypothesis #3: Our innovative SF1/U2AF65/U2AF35/RNA preparation positions us to locate the protein and RNA subunits in the complex, and to test the structural and functional effects of U2AF35 mutations that frequently cause myelodysplasia, hematological malignancies and lung cancer. Our overall approach entails a multi-front attack on all aims using a multidisciplinary strategy. Our core biophysical technologies entail X-ray crystallography, fluorescence anisotropy, isothermal titration calorimetry, and small-angle X-ray scattering with purified proteins. To meet challenges and propel the field in new directions, we will utilize a powerful combination of innovative methods including protein and RNA labeling, Forster resonance energy transfer, small-angle neutron scattering, and site-specific photo-crosslinking followed by LC-MS/MS. These tools are complemented by strong collaborations to study splicing factor functions in nuclear extracts and in living cells. Our research is grounded in the fundamental structure and function of 3' splice site recognition yet will broadly impact the field's understanding of aberran splicing, which is a dominant cause of blood disorders, neuromuscular diseases, leukemias and cancers.